Fig. 4

Zscan4 expression is induced by DNA damage and regulated by the ATM/TRAF6/TAK1/p65 signaling axis. a PSC27 cells were treated by bleomycin (50 μg/ml) with or without KU55933 (KU, 10 μM) in culture. Cell lysates were immunoprecipitated with anti-p-ATM, with the immunoprecipitates (IPs) analyzed with anti-p-ATM and anti-TRAF6, respectively. b PSC27 cells lentivirally infected with scramble or TRAF6-specific shRNAs were treated with bleomycin, with lysates collected at indicated time points and subject to IP and immunoblot assays. c Anti-TAK1-based IP of PSC27 cells treated with bleomycin in the presence or absence of 5Z-7 (500 nM), followed by immunoblot analysis. d Primary PSC27 cells or those stably expressing shRNA to TRAF6 were treated with bleomycin, with IPs pulled down by anti-TRAF6 and examined by immunoblots. e Cytoplasmic and nuclear protein samples from control and bleomycin-treated PSC27 cells were analyzed for TAK1 activation and NF-κB nuclear translocation. GAPDH and Histone H1, cytoplasmic and nuclear loading controls, respectively. f Chromatin immunoprecipitation (ChIP) was performed to identify potential NF-κB binding sites in Zscan4 proximal promoter. Zscan4-p1/p2/p3 denotes 3 representative genomic sites in promoter region, with known NF-κB binding sites from WNT16B, SFRP2, IL-6, and IL-8 selected as positive controls. g Zscan4 transcript expression in PSC27 cells stably expressing an NF-κB-null mutant and treated by bleomycin, mitoxantrone or radiation. Signals normalized to untreated cells. h Expression profiling of typical ASAP factors (IL-6/Timp-1) and Zscan4 in stromal cells exposed to bleomycin. Left (histograms), cells were pretreated with inhibitors of NF-κB, ATM, or TAK1 (Bay, KU or 5Z-7, respectively) before addition of bleomycin, with transcripts collected for analysis 24 h after genotoxic treatment (data normalized to untreated sample per factor set). Right (immunoblots), protein level assessment of IL-6, Timp-1, and Zscan4 24 h after cell exposure to bleomycin. GAPDH, loading control. Data in all bar plots are shown as mean ± SD and representative of 3 biological replicates. BLEO bleomycin, KU KU55933, 5Z-7 5Z-7-oxozeaenol, Bay Bay 11–7082. Data in g, h were analyzed by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ^P > 0.05